Plasmid construction and sgRNA design
Cas9 coding region of pX330 plasmid (Gifted from Dr. Feng Zhang, Addgene accession no. 42230) was replaced with EGFP cDNA (Fig. 1B). EGFP sequence was PCR amplified from pEGFP-N1 (Clontech cat# 6059-1) (Fig. 1C) using following primers: EGFP-F: 5’-GGCCACCGGT GATCCACCGGTCGCCACCAT-3’ (20bp) and EGFP-R: 5’- GGCCGAATTC TTACTTGTACAGCTCGTCCATG-3’ (22bp) with AgeI site at 5’-end and EcoRI site at 3’-end (AgeI and EcoRIare shown by underline). PCR was performed at 94°C for 4min, 24 cycles of 94°C for 30s, 56°C for 30s, 72°C for 1min and 72°C for 10min. EGFP PCR products were digested with AgeI and EcoRI (NEB) and inserted into AgeI and EcoRI sites of pX330. Resultant pX330-EGFP plasmid (Fig. 1B) was confirmed by sequencing. Oligos coding for sgRNA targets were synthesized by Genewiz (Beijing, China), annealed at 95°C for 5min and ramped down to 25°C (-5°C/min) and then subcloned into BbsI sites of pX330-EGFP. BE4 plasmid (Fig. 1A) was gifted from David Liu lab (Addgene access no. 100802). The sgRNAs were designed using online platform https://benchling.com/ and all sgRNAs oligos are listed in Supplementary Table 1.